DESCRIPTION: In cerebellar granule cells, metabotropic glutamate receptors (mGluRs) elevate intracellular Ca2+ by increasing Ca2+ influx across the cell membrane in addition to releasing this cation from intracellular stores via an increase in inositol triphosphate. Several lines of evidence indicate that activation or facilitation of Ca2+ entry by mGluR1 is coupled to the activity of ryanodine-sensitive Ca2+ stores. Investigations of the ability of the mGluR1 agonist, t-ACPD (1-aminocyclopentane-trans-1,3-dicarboxylic acid) to facilitate or increase a dihydropyridine-sensitive L-type calcium current in cerebellar granular cells have lead to the following observations. The facilitation was specific for group 1 mGluRs and selective for L-type currents. In intact cells the facilitation developed and was reversed in seconds after addition and removal of agonist. It was not blocked by the PKA inhibitory peptide, PKI, it did not require increases in IP3 and it was not blocked by heparin. Therefore, cAMP, IP3 and PKC pathways did not appear to be involved. Dialysis of the cell with 20 mM BAPTA also failed to block the increased in current, ruling out the involvement of global changes in cell calcium, but not perhaps local changes in a buffer-inaccessible intracellular compartment. A striking feature of the mGluR1-mediated increase in current was that it exhibited oscillations in amplitude during exposure to the agonist. The period between depolarizations should have been sufficient to prevent build-up and removal of voltage-dependent inactivation, hence this does not appear to underlie the oscillatory changes in current amplitude. Several observations indicate the involvement of ryanodine receptors in this phenomenon: Current oscillations can be initiated by caffeine and ryanodine and ruthenium red block current oscillations initiated by either caffeine or t-ACPD. Importantly, ryanodine blocks only the oscillations and not the current itself and the onset of ryanodine blockade was accelerated by increasing the frequency of cell depolarizations. Finally the facilitated current oscillations initiated by either caffeine or t-ACPD continue after excision of inside-out patches and unlike intact cells after removal of agonist. Ryanodine blocks the current oscillations in excised patches. Based on these observations and on some similarities between the behavior of a granule cell and skeletal muscle L-type calcium currents. the investigator has proposed a model involving close physical interactions between a ryanodine-sensitive calcium store and a L-type calcium channel in the plasma membrane, similar at least in concept to that thought to exist in skeletal muscle. In the stimulated granule cell, this association leads to a localized oscillator circuit involving the release of calcium from the ryanodine sensitive store and calcium entry via the L-type calcium current. The experiments in this proposal will investigate the mechanism of mGluR1 signal transduction involved in the activation of Ca2+ influx and the mechanisms by which intracellular Ca2+ stores control Ca2+ entry. Four specific Aims are proposed. The first Aim will investigate the role of receptor-dependent processes in the activation of Ca2+ influx by mGluRs and determine whether signaling involves a direct action of a G protein, known G protein-coupled effectors, or a novel second messenger. The second Aim seeks to identify the retrograde signal produced by the ryanodine receptor that is responsible for increasing Ca2+ channel activity in patches of membrane. Aim-3 will characterize the spatial distributions of RyRs and L-type channels and determine whether the ER and plasma membranes form specialized junctions in regions where the proteins co-localize. The fourth Aim involves characterizations of the Ca2+i pools mobilized by mGluR1 and muscarinic receptors in granule cells and determine the contribution of store-coupled Ca2+ influx to electrical excitability.